Negative Elongation Factor-Mediated Suppression of RNA Polymerase II Elongation of Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene Expression

In primary effusion lymphoma (PEL) cells infected with latent Kaposi's sarcoma-associated herpesvirus (KSHV), the promoter of the viral lytic switch gene, Rta, is organized into bivalent chromatin, similar to cellular developmental switch genes. Histone deacetylase (HDAC) inhibitors (HDACis) reactivate latent KSHV and dramatically remodel the viral genome topology and chromatin architecture. However, reactivation is not uniform across a population of infected cells. We sought to identify an HDACi cocktail that would uniformly reactivate KSHV and reveal the regulatory HDACs. Using HDACis with various specificities, we found that class I HDACis were sufficient to reactivate the virus but differed in potency. Valproic acid (VPA) was the most effective HDACi, inducing lytic cycle gene expression in 75% of cells, while trichostatin A (TSA) induced less widespread lytic gene expression and inhibited VPA-stimulated reactivation. VPA was only slightly superior to TSA in inducing histone acetylation of Rta's promoter, but only VPA induced significant production of infectious virus, suggesting that HDAC regulation after Rta expression has a dramatic effect on reactivation progression. Ectopic HDACs 1, 3, and 6 inhibited TPA-stimulated KSHV reactivation. Surprisingly, ectopic HDACs 1 and 6 stimulated reactivation independently, suggesting that the stoichiometries of HDAC complexes are critical for the switch. Tubacin, a specific inhibitor of the ubiquitin-binding, proautophagic HDAC6, also inhibited VPA-stimulated reactivation. Immunofluorescence indicated that HDAC6 is expressed diffusely throughout latently infected cells, but its expression level and nuclear localization is increased during reactivation. Overall, our data suggest that inhibition of HDAC classes I and IIa and maintenance of HDAC6 (IIb) activity are required for optimal KSHV reactivation.

Section: Discussionmentioning

confidence: 98%

Histone Deacetylase Classes I and II Regulate Kaposi's Sarcoma-Associated Herpesvirus Reactivation

Shin

DeCotiis

Giron

et al. 2014

“…Alternatively, it is possible that pausing occurs during viral transcription but the paused Pol II is released quickly and therefore is not captured by Pol II ChIP-seq. The roles of DSIF and NELF in transcription of HSV-1 genes have not been reported, although these factors help control the transcription of other herpesviruses (Kaposi's sarcoma-associated herpesvirus [KSHV] and EpsteinBarr virus [EBV] [50,51]). …”

Section: Discussionmentioning

confidence: 99%

Infection by Herpes Simplex Virus 1 Causes Near-Complete Loss of RNA Polymerase II Occupancy on the Host Cell Genome

Abrisch

Eidem

Yakovchuk

et al. 2016

Lytic infection by herpes simplex virus 1 (HSV-1) triggers a change in many host cell programs as the virus strives to express its own genes and replicate. Part of this process is repression of host cell transcription by RNA polymerase II (Pol II), which also transcribes the viral genome. Here, we describe a global characterization of Pol II occupancy on the viral and host genomes in response to HSV-1 infection using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). The data reveal near-complete loss of Pol II occupancy throughout host cell mRNA genes, in both their bodies and promoter-proximal regions. Increases in Pol II occupancy of host cell genes, which would be consistent with robust transcriptional activation, were not observed. HSV-1 infection induced a more potent and widespread repression of Pol II occupancy than did heat shock, another cellular stress that widely represses transcription. Concomitant with the loss of host genome Pol II occupancy, we observed Pol II covering the HSV-1 genome, reflecting a high level of viral gene transcription. Interestingly, the positions of the peaks of Pol II occupancy at HSV-1 and host cell promoters were different. The primary peak of Pol II occupancy at HSV-1 genes is ϳ170 bp upstream of where it is positioned at host cell genes, suggesting that specific steps in transcription are regulated differently at HSV-1 genes than at host cell mRNA genes. IMPORTANCE We investigated the effect of herpes simplex virus 1 (HSV-1) infection on transcription of host cell and viral genes by RNA polymerase II (Pol II). The approach we used was to determine how levels of genome-bound Pol II changed after HSV-1 infection.We found that HSV-1 caused a profound loss of Pol II occupancy across the host cell genome. Increases in Pol II occupancy were not observed, showing that no host genes were activated after infection. In contrast, Pol II occupied the entire HSV-1 genome. Moreover, the pattern of Pol II at HSV-1 genes differed from that on host cell genes, suggesting a unique mode of viral gene transcription. These studies provide new insight into how HSV-1 causes changes in the cellular program of gene expression and how the virus coopts host Pol II for its own use. Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that proliferates in the nuclei of host cells during lytic infection (reviewed in reference 1). HSV-1 can cause lifelong infection by establishing asymptomatic latency in host sensory neurons, where it remains in a transcriptionally silenced state until it is stimulated by stress to reactivate and replicate in epithelial cells (2). The majority of the population are infected with HSV-1, which is largely responsible for oral cold sores; however, in rarer cases, it can cause severe conditions, such as blindness and encephalitis (3, 4). Recent experimental and epidemiological evidence also suggests a role for recurrent HSV-1 infection in Alzheimer's disease (5). Despite its clear medical significance, the relationship between the virus ...

“…9). Since this effect was observed in iSLK cells infected with Rta null virus, in which Rta expression is induced by Dox from an ectopic cassette carried in the cellular genome (42,43), it proves that Pin1 does indeed interact with Rta protein posttranslationally in infected cells, rather than solely induce Rta expression during reactivation. We showed that Pin1 cooperated with Rta to transactivate DE genes (Fig.…”

Section: Discussionmentioning

confidence: 96%

“…A doxycycline (Dox)-inducible Rta cell line TREx BCBL-1-Rta (a gift of J. Jung, University of Southern California [USC] [38]), a wild-type, KSHV-infected B lymphoma cell line BCBL-1, and uninfected B lymphoma cell line BL-41 were maintained in RPMI 1640 supplemented with 12% fetal bovine serum (Sigma) as previously described (15,22 Vieira), were maintained as previously described (15,22,(39)(40)(41)(42)(43)(44). iSLK cell lines contain Dox-inducible Rta and were gifts of D. Ganem (42).…”

Section: Methodsmentioning

confidence: 99%

“…independent of Pin1. Thus, we employed an iSLK-BAC16-based system, in which the viral allele of Rta is rendered defective by insertion of a stop codon (BAC16 Rta-stop) and functionally replaced with a Dox-inducible allele of Rta integrated in the SLK cellular genome (43). We measured intracellular vDNA by qPCR in cells that were treated with Dox and/or juglone or not treated with Dox and/or juglone.…”

Section: Pin1 Relocalizes Rta In Vivo To Analyze Interactions Of Pinmentioning

confidence: 99%

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The Cellular Peptidyl-Prolyl cis / trans Isomerase Pin1 Regulates Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency

Guito

Gavina

Palmeri

et al. 2014

Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma and primary effusion lymphoma. KSHV-infected cells are predominantly latent, with a subset undergoing lytic reactivation. Rta is the essential lytic switch protein that reactivates virus by forming transactivation-competent complexes with the Notch effector protein RBP-Jk and promoter DNA. Strikingly, Rta homolog analysis reveals that prolines constitute 17% of conserved residues. Rta is also highly phosphorylated in vivo. We previously demonstrated that proline content determines Rta homotetramerization and function. We hypothesize that prolinedirected modifications regulate Rta function by controlling binding to peptidyl-prolyl cis/trans isomerases (PPIases). Cellular PPIase Pin1 binds specifically to phosphoserine-or phosphothreonine-proline (pS/T-P) motifs in target proteins. Pin1 dysregulation is implicated in myriad human cancers and can be subverted by viruses. Our data show that KSHV Rta protein contains potential pS/T-P motifs and binds directly to Pin1. Rta transactivation is enhanced by Pin1 at two delayed early viral promoters in uninfected cells. Pin1's effect, however, suggests a rheostat-like influence on Rta function. We show that in infected cells, endogenous Pin1 is active during reactivation and enhances Rta-dependent early protein expression induced by multiple signals, as well as DNA replication. Surprisingly, ablation of Pin1 activity by the chemical juglone or dominant-negative Pin1 enhanced late gene expression and production of infectious virus, while ectopic Pin1 showed inhibitory effects. Our data thus suggest that Pin1 is a unique, dose-dependent molecular timer that enhances Rta protein function, but inhibits late gene synthesis and virion production, during KSHV lytic reactivation.